CONICET | Buscador de Institutos y Recursos Humanos

Capacitation is a process that prepares mammalian sperm to undergo an exocytotic event called acrosome reaction (AR) which in turn, is an essential step of fertilization. The study and characterization of the proteins involved in these events is extremely important in order to understand the dynamics of the whole process. In the present work we evaluated the role of Valosin Containing Protein (VCP/p97) in mouse sperm. We found that VCP is localized in the equatorial segment and along the flagellum. In addition, we observed that VCP is cleaved and released during AR. In contrast to human sperm, VCP is not phosphorylated in tyrosine residues. To elucidate how VCP is involved in the capacitation process we used a pharmacological approach. Mouse sperm were incubated in capacitating conditions with or without VCP inhibitors. Several aspects of the capacitation such as phosphorylation of PKA substrates, tyrosine phosphorylation, AR or motility were evaluated. In these experiments, we used four VCP inhibitors: NMS-873, DBeq, CB-5083 and ML-240. By Western blot, we observed no significant differences in the levels of phosphorylation of PKA substrates. Surprisingly, we noticed that all four inhibitors completely abolished tyrosine phosphorylation although this inhibition could be bypassed by using cAMP analogs. Next, we evaluated AR using transgenic EGFP sperm and flow cytometry. We observed that the AR induced by progesterone is strongly inhibited by NMS-873. Finally, we study sperm motility using CASA with different concentrations of this inhibitor and in neither of these, the motility was significantly changed. Taken together, these results indicate that VCP plays an important role in mouse sperm capacitation and if inhibited, these cells cannot undergo AR. On the other hand, motility does not appear to be modified by VCP inhibition.